Methods And Compositions For Gene Inactivation

ABSTRACT

Disclosed herein are methods and compositions for inactivating CCR-5 genes, using zinc finger nucleases (ZFNs) comprising a zinc finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding ZFNs, vectors comprising polynucleotides encoding ZFNs, such as adenovirus (Ad) vectors, and cells comprising polynucleotides encoding ZFNs and/or cells comprising ZFNs are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/805,707, filed May 23, 2007, which claims the benefit ofU.S. Provisional Application No. 60/808,501, filed May 25, 2006; U.S.Provisional Application No. 60/847,269, filed Sep. 26, 2006 and U.S.Provisional Application No. 60/926,911, filed Apr. 30, 2007, all ofwhich disclosures are hereby incorporated by reference in theirentireties herein.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

TECHNICAL FIELD

The present disclosure is in the fields of polypeptide and genomeengineering and homologous recombination.

BACKGROUND

Various methods and compositions for targeted cleavage of genomic DNAhave been described. Such targeted cleavage events can be used, forexample, to induce targeted mutagenesis, induce targeted deletions ofcellular DNA sequences, and facilitate targeted recombination at apredetermined chromosomal locus. See, for example, United States PatentPublications 20030232410; 20050208489; 20050026157; 20050064474;20060188987; and International Patent Publication WO 07/014,275, thedisclosures of which are incorporated by reference in their entiretiesfor all purposes.

CCR5, a 7-transmembrane chemokine receptor, is the major co-receptor forHIV-1 entry into CD4 T cells (Samson et al. (1996) Nature 382:722-725;Deng et al. (1996) Nature 381:661-666; Alkhatib (1996) Science272:1955-1958). Since the discovery of the HIV-1 resistance conferringhomozygous Δ32 deletion in the CCR5 gene, CCR5 has been intensivelystudied as a prime target for HIV therapy. Although small molecules havebeen shown to induce receptor internalization or block CCR5-HIVinteraction (Fatkenheuer et al. (2005) Nat. Med. 11:1170-1172), thesesmall molecule approaches have resulted in the development of resistancevia selection for escape mutants which interestingly continue to useCCR5 for viral entry (Kuhmann et al. (2004) J. Virol. 78:2790-2807).Similarly, intrabody, antisense and RNAi-based approaches have to dateonly partially blocked CCR5 expression.

Thus, there remains a need for compositions that completely knock-outCCR5 for phenotypic penetrance and long-term resistance to HIVinfection.

SUMMARY

Disclosed herein are compositions and methods for partial or completeinactivation of a target gene. Also disclosed are methods of making andusing these compositions (reagents), for example to inactivate a gene ina cell for therapeutic purposes and/or to produce cell lines in which atarget gene is inactivated.

In one aspect, provided herein are zinc finger nucleases (ZFNs) thathave target sites in the human CCR-5 gene. In some embodiments, cleavagewithin the CCR-5 gene with these nucleases results in permanentdisruption (e.g., mutation) of the CCR5 gene. In certain embodiments,the zinc finger domain(s) is(are) engineered to bind to a target siteupstream of the naturally occurring CCR5 Δ32 mutation. The zinc fingerproteins may include 1, 2, 3, 4, 5, 6 or more zinc fingers, each zincfinger having a recognition helix that binds to a target subsite in thetarget gene. In certain embodiments, the target gene is CCR-5 and thezinc finger proteins comprise 4 fingers (designated F1, F2, F3 and F4and ordered F1 to F4 from N-terminus to C-terminus) and comprise theamino acid sequence of the recognition regions shown in Table 1.

Thus, in certain aspects, provided herein is a protein comprising anengineered zinc finger protein DNA-binding domain, wherein theDNA-binding domain comprises four zinc finger recognition regionsordered F1 to F4 from N-terminus to C-terminus, and wherein F1, F3, andF4 comprise the following amino acid sequences: DRSNLSR (SEQ ID NO:2);F3: RSDNLAR (SEQ ID NO:4); and F4: TSGNLTR (SEQ ID NO:8). In certainembodiments, F2 comprises the amino acid sequence ISSNLNS (SEQ ID NO:5).Alternatively, F2 comprises the amino acid sequence VSSNLTS (SEQ IDNO:6).

Any of the proteins described herein may further comprise a cleavagedomain and/or a cleavage half-domain (e.g., a wild-type or engineeredFokI cleavage half-domain). Thus, in any of the ZFNs described herein,the nuclease domain may comprise a wild-type nuclease domain or nucleasehalf-domain (e.g., a FokI cleavage half domain). In other embodiments,the ZFNs comprise engineered nuclease domains or half-domains, forexample engineered FokI cleavage half domains that form obligateheterodimers. See, e.g., U.S. Provisional Patent Application No.60/808,486, filed May 25, 2006.

In another aspect, the disclosure provides a polynucleotide encoding anyof the proteins described herein. Any of the polynucleotides describedherein may also comprise sequences (donor or patch sequences) fortargeted insertion into the target gene (e.g., CCR-5).

In yet another aspect, a gene delivery vector comprising any of thepolynucleotides described herein is provided. In certain embodiments,the vector is an adenovirus vector (e.g., an Ad5/35 vector). Thus, alsoprovided herein are adenovirus (Ad) vectors comprising a sequenceencoding at least one zinc finger nuclease (ZFN) and/or a donor sequencefor targeted integration into a target gene. In certain embodiments, theAd vector is a chimeric Ad vector, for example an Ad5/35 vector. Inadditional embodiments, the target gene is the human CCR-5 gene. Thevectors described herein may comprise donor sequences. In certainembodiments, a single vector comprises sequences encoding one or moreZFNs and the donor sequence(s). In other embodiments, the donorsequence(s) are contained in a first vector and the ZFN-encodingsequences are present in a second vector.

The ZFN-sequences of the vectors (e.g., Ad vectors) described hereinwill typically encode a fusion of a zinc finger protein (ZFP) and acleavage domain or cleavage half-domain (i.e., a nuclease domain). Thezinc finger protein portion of the ZFN is engineered to bind to a targetsite in the target gene. Zinc finger proteins may include 1, 2, 3, 4, 5,6 or more zinc fingers, each zinc finger having a recognition helix thatbinds to a target subsite in the target gene. In certain embodiments,the target gene is CCR-5 and the zinc finger proteins comprise 4 fingers(designated F1, F2, F3 and F4) and comprise the amino acid sequence ofthe recognition regions shown in Table 1.

In any of the polynucleotides or proteins described herein, the cleavagedomain may comprise at least one cleavage domain or at least onecleavage half-domain. In certain embodiments, the cleavage domain orcleavage half-domain is a wild-type cleavage domain (e.g., a FokIwild-type cleavage half-domain). In other embodiments, the cleavagedomain or cleavage half-domain is engineered.

In yet another aspect, the disclosure provides an isolated cellcomprising any of the proteins, polynucleotides and/or vectors describedherein. In certain embodiments, the cell is selected from the groupconsisting of a hematopoietic stem cell, a T-cell (e.g., CD4⁺ T-cell), amacrophage, a dendritic cell and an antigen-presenting cell. In anotheraspect, cells comprising one or more Ad vectors as described herein(Ad-ZFN, Ad-ZFN-donor and/or Ad-donor vectors) are also described. Cellsinclude, for example, peripheral Blood Mononuclear Cells (PBMCs),macrophages, mesenchymal stem cells, human embryonic stem cells (hEScells), hematopoietic stem cell (e.g., CD34⁺ cells), T-cells (e.g., CD4⁺cells), dendritic cells or antigen-presenting cells; or a cell line suchas K562 (chronic myelogenous leukemia), HEK293 (embryonic kidney), PM-1(CD4⁺ T-cell), THP-1 (monocytic leukemia) or GHOST (osteosarcoma).

In another aspect, described herein are methods of inactivating a targetgene in a cell by introducing one or more proteins, polynucleotidesand/or vectors into the cell as described herein. In any of the methodsdescribed herein the ZFNs may induce targeted mutagenesis, targeteddeletions of cellular DNA sequences, and/or facilitate targetedrecombination at a predetermined chromosomal locus. Thus, in certainembodiments, the ZFNs delete one or more nucleotides of the target gene.In other embodiments, a genomic sequence in the target gene is replaced,for example using an Ad-ZFN as described herein and a “donor” sequencethat is inserted into the gene following targeted cleavage with the ZFN.The donor sequence may be present in the Ad-ZFN vector, present in aseparate Ad vector or, alternatively, may be introduced into the cellusing a different nucleic acid delivery mechanism. In certainembodiments, the target gene is a CCR-5 gene.

In another aspect, methods of using the zinc finger proteins and fusionsthereof for mutating the CCR-5 gene and/or inactivating CCR-5 functionin a cell or cell line are provided. Thus, a method for inactivating aCCR-5 gene in a human cell is provided, the method comprisingadministering to the cell any of the proteins or polynucleotidesdescribed herein.

In yet another aspect, the disclosure provides a method for treating orpreventing HIV infection in a subject, the method comprising: (a)introducing, into a cell, a first nucleic acid encoding a firstpolypeptide, wherein the first polypeptide comprises: (i) a zinc fingerDNA-binding domain that is engineered to bind to a first target site inthe CCR5 gene; and (ii) a cleavage domain; under conditions such thatthe polypeptide is expressed in the cell, whereby the polypeptide bindsto the target site and cleaves the CCR5 gene; and (b) introducing thecell into the subject. In certain embodiments, the cell is selected fromthe group consisting of a hematopoietic stem cell, a T-cell, amacrophage; a dendritic cell and an antigen-presenting cell. The nucleicacid may comprise any of the polynucleotides described herein. In any ofthe methods, the first nucleic acid may further encode a secondpolypeptide, wherein the second polypeptide comprises: (i) a zinc fingerDNA-binding domain that is engineered to bind to a second target site inthe CCR5 gene; and (ii) a cleavage domain; such that the secondpolypeptide is expressed in the cell, whereby the first and secondpolypeptides bind to their respective target sites and cleave the CCR5gene. Similarly, any of these methods may further comprise the step ofintroducing into the cell a second nucleic acid, wherein the secondnucleic acid contains two regions of homology to the CCR-5 gene,flanking a sequence that is non-homologous to the CCR-5 gene.

In any of the methods and compositions described herein, the cell canbe, for example, a hematopoietic stem cell (e.g., a CD34⁺ cell), aT-cell (e.g., a CD4⁺ cell), a macrophage, a dendritic cell or anantigen-presenting cell; or a cell line such as K562 (chronicmyelogenous leukemia), HEK293 (embryonic kidney), PM-1 (CD4⁺ T-cell),THP-1 (monocytic leukemia) or GHOST (osteosarcoma).

Furthermore, any of the methods described herein can be practiced invitro, in vivo and/or ex vivo. In certain embodiments, the methods arepracticed ex vivo, for example to modify PBMCs, e.g., T-cells, to makethem resistant to HIV infection via disruption of CCR-5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematic diagrams of the Ad5/35 vectors in which sequencesencoding E1 are deleted and replaced with a transgene expressioncassette (e.g., encoding GFP, ZFNs and/or donor sequences).

FIG. 2 shows the amino acid sequence of the wild-type FokI cleavagehalf-domain (SEQ ID NO:33). Positions at which the sequence can bealtered (486, 490, 499 and 538) to form engineered cleavage half-domainsare bolded and underlined.

FIG. 3 shows the amino acid sequence of an exemplary engineered cleavagehalf-domain (SEQ ID NO:34) that forms a heterodimer with the engineeredcleavage half-domain shown in FIG. 4. Positions at which the sequencewas altered as compared to wild-type (corresponding to amino acidresidues 486 and 499) are underlined.

FIG. 4 shows the amino acid sequence of another exemplary engineeredcleavage half-domain (SEQ ID NO:35) that can be used in the ZFNsdescribed herein. Positions at which the sequence was altered ascompared to wild-type (corresponding to amino acid residues 490 and 538)are underlined.

FIG. 5 shows the nucleotide sequence of portion of a CCR-5 gene (SEQ IDNO:36) used to make a donor (patch) sequence having CCR-5 homology arms.See also Example 1.

FIG. 6 shows the nucleotide sequence of a 47 bp “patch” sequence (SEQ IDNO:37) used for insertion into the CCR-5 gene. See also Example 1.

FIG. 7 shows the nucleotide sequence of the donor sequence (SEQ IDNO:38) used for targeted insertion into the CCR-5 gene. The 5′ CCR-5homology arm corresponds to nucleotides 1-471; the “patch” sequence fortargeted insertion into CCR-5 is underlined and corresponds tonucleotides 472-518; and the 3′ CCR-5 homology arm corresponds tonucleotides 519-1928. See also Example 1.

FIG. 8 depicts sequences (SEQ ID NOS: 54-66) of a portion of the CCR-5gene in cells transduced with Ad5/35-ZFN. Cell type is shown in Column3. Missing bases as compared to wild-type CCR-5 sequence are denotedwith a period.

FIG. 9 (SEQ ID NOS: 43, 44, 46-50) depicts sequence analysis of aportion of the CCR-5 gene in cells transduced with an Ad5/35-ZFN. Celltype is indicated in Column 3. The modified genomes shown in this figurehad various small insertions (underlined bases) in the CCR-5 gene and,in one case, a deletion, indicated by a period.

FIG. 10 (SEQ ID NOS: 51-53) depicts sequence analysis of a portion ofthe CCR-5 gene in cells transduced with an Ad5/35-ZFN. The modifiedgenomes shown in this Figure had various longer insertions (underlinedbases) in the CCR-5 gene.

FIG. 11, panels A and B, depict the time course of percentage of CCR-5modified T-cells in T-cells transduced with Ad5/35 ZFN215 (Panel A) andAd5/35 ZFN 224 (Panel B), following challenge with wild-type HIV or mockinfection.

FIG. 12 (SEQ ID NOS: 67 and 68) is a schematic depicting the targetsites in the CCR5 gene for CCR5-ZFNs pairs 215 and 224.

FIG. 13 shows levels of target gene disruption in GHOST-CCR5 cellstransduced with an Ad5/35 vector encoding the indicated ZFNs targetingeither CCR5 or IL2-Rγ. See Example 14. Lower migrating products(indicated by arrows) are a direct measure of ZFN-mediated genedisruption. “NTD” indicates non-transduced cells.

FIG. 14, panels A and B, are graphs depicting flow cytometrymeasurements of CCR5 surface expression (FIG. 14A) or GFP expression(FIG. 14B) of GHOST-CCR5 cells transduced with an Ad5/35 vector encodingZFN 215 or ZFN 224. FIG. 14A depicts decreased CCR5 surface expressionas measured by flow cytometry in GHOST-CCR5 cells transduced with theindicated vector. NTD refers to non-transduced; IL2R refers to cellscontaining IL2Rγ-targeted ZFNs; 215 and 224 refer to cells containingZFN pair 215 or 224, respectively. “MFI” indicates mean fluorescenceintensity. FIG. 14B shows protection from challenge with HIV-1_(BAL) asmeasured by flow cytometry 48 hours after HIV challenge of CCR5-ZFN215and CCCR-ZFN224 modified cells compared to IL-2rγ ZFN and controlGHOST-CCR cells. GFP fluorescence indicates HIV entry and is plotted asan average percent infected relative to positive control. Bar graphsrepresent averages of triplicates.

FIG. 15 shows the level of ZFN-disrupted CCR alleles, determined byCel-1 assay, at days 3, 10, 21, 31, 42 and 52 post-HIV-1 challenge withR5-tropic HIV-1_(BAL) or after mock HIV infection. Cells with disruptedCCR5 alleles remained at stable levels in mock infected cultures, butwere enriched in the presence of HIV-1.

FIG. 16 shows sequences of CCR5 alleles in ZFN-treated PM1 cells at day52 post-HIV challenge.

FIG. 17 shows levels of ZFN-disrupted CCR5 alleles in primary CD4 Tcells from an anonymous healthy donor transduced with an Ad5/35 vectorexpressing CCR5-ZFN215, CCR5-ZFN224, or GFP; at MOIs of 30 or 100, asdetermined by Surveyor™ nuclease assay. Bands corresponding to disruptedCCR5 alleles are indicated by arrows. The percentage of disrupted CCR5alleles is indicated below each lane.

FIG. 18 depicts the population doubling rate for CD4 T cells transducedwith Ad5/35 vectors whose genomes encoded either CCR5-ZFNs or GFP(control cells). Cells were transduced with the Ad5/35 vectors on day 0.The line connecting points shown by triangles depicts doubling rates ofnon-transduced cells; the line connecting points shown by squaresdepicts doubling rates of Ad5/35 CCR5 ZFN 224-transduced cells; and theline connecting points shown by diamonds depicts doubling rates ofAd5/35 GFP transduced cells.

FIG. 19 depicts enrichment of ZFN-disrupted CCR5 alleles in ZFN215-transduced CD4⁺ T cells over time following in vitro challenge withCCR5-tropic HIV-1_(US1), compared to mock infected cultures. CCR5disruption was measured using the Surveyor® nuclease (Cel-1) assay. Theline joining squares depicts HIV infected cells and the line joining thetriangles depicts mock infected cells. An ˜10% starting level ofZFN-disrupted CCR5 alleles was obtained by mixing Ad5/35 transduced CD4T cells with unmodified CD4 T cells (1:3).

FIG. 20 is a graph depicting average intranuclear P53BP1 immunostainingfoci in primary CD4⁺ T Cells, determined 24 hours after transductionwith Ad5/35 vectors expressing CCR5 ZFN pairs 215 or 224. Intranuclearfoci were counted from a minimum of 100 nuclei per condition usingVolocity™ software. Results obtained from positive control cells treatedwith etoposide and negative control cells (non-transduced) are alsoshown.

FIG. 21 is a graph depicting in vivo CCR5 disruption frequencies,measured using the Surveyor® nuclease (Cel-1) assay, in CD4 cellsisolated on day 40 from the spleens of control (mock infected) orHIV-infected mice. Results for each group were averaged and analyzedusing an unpaired T-test.

DETAILED DESCRIPTION

Disclosed herein are zinc finger nuclease (ZFNs) targeting the humanCCR5 gene (CCR5-ZFNs). These ZFNs efficiently generate a double strandbreak (DSB), for example at a predetermined site in the CCR5 codingregion. The site can be, for example, upstream of the CCR5Δ32 mutation.Transient expression of the ZFNs described herein promotes highlyefficient and permanent disruption of the CCR5 gene in human cells,including primary human CD4 T lymphocytes, confers robust protectionagainst HIV-1 infection and provides a powerful selective advantage tothese cells both in vitro and in vivo.

In particular, transient delivery of CCR5-ZFNs results in the permanentdisruption of the human CCR5 gene with efficiencies surpassing 50% inprimary human CD4 T cells. CCR5-ZFN action is highly specific and welltolerated, as revealed by (i) examination of the stability, growth andengraftment characteristics of the ZFN-modified sub-population even inthe absence of selection, (ii) direct staining for intranuclearDSB-induced 53BP1 foci, and (iii) testing for cleavage at the mostsimilar putative off-target genomic sites. Moreover, in the presence ofa selective pressure in the form of active HIV-1 infection,ZFN-modification confers a profound survival advantage duringCCR5-tropic (but not CXCR4-tropic) HIV-1 challenge assays in vitro tolevels comparable to those obtained with homozygous CCR5Δ32 cells.

CCR5-ZFN-mediated genome editing as described herein may be employed togenerate a CCR5 null genotype in primary human cells. Moreover, asexpected for a genetically determined trait, the ZFN-modified cellsdemonstrated stable and heritable resistance to HIV-1 infection both invitro and in vivo.

Small molecule, intrabody, and anti-sense or RNAi-based approaches toHIV treatment via CCR5 disruption incompletely repress or block CCR5 atthe mRNA or protein level. See, Levine et al. (2006) Proc. Nat'l Acad.Sci. USA 103:17372-17377; Trkola et al. (2002) Proc. Nat'l Acad. Sci.USA 99:395-400). Thus, unlike other approaches, the CCR5-ZFNs describedherein generate a true CCR5 null cell, which, like the naturallyselected CCR5Δ32, is permanently and completely CCR5 negative,preferentially survives HIV-1 infection, and gives rise to daughtercells that are equally resilient to HIV-1 infection. Permanent geneticmodification by CCR5-ZFNs blocks viral entry without the requirement forthe integration of any foreign DNA into the genome, as transient ZFNgene delivery and expression is sufficient to eliminate CCR5 expression.

Also disclosed herein are adenovirus (Ad) vectors comprising ZFNs and/ordonor sequences and cells comprising these Ad vectors. These Ad vectorsare useful in methods for targeted cleavage of cellular chromatin andfor targeted alteration of a cellular nucleotide sequence, e.g., bytargeted cleavage followed by non-homologous end joining or by targetedcleavage followed by homologous recombination between an exogenouspolynucleotide (comprising one or more regions of homology with thecellular nucleotide sequence) and a genomic sequence.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

DEFINITIONS

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

Zinc finger binding domains can be “engineered” to bind to apredetermined nucleotide sequence. Non-limiting examples of methods forengineering zinc finger proteins are design and selection. A designedzinc finger protein is a protein not occurring in nature whosedesign/composition results principally from rational criteria. Rationalcriteria for design include application of substitution rules andcomputerized algorithms for processing information in a database storinginformation of existing ZFP designs and binding data. See, for example,U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

A “selected” zinc finger protein is a protein not found in nature whoseproduction results primarily from an empirical process such as phagedisplay, interaction trap or hybrid selection. See e.g., U.S. Pat. No.5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988; U.S. Pat.No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO 96/06166; WO98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197 and WO02/099084.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

A “homologous, non-identical sequence” refers to a first sequence whichshares a degree of sequence identity with a second sequence, but whosesequence is not identical to that of the second sequence. For example, apolynucleotide comprising the wild-type sequence of a mutant gene ishomologous and non-identical to the sequence of the mutant gene. Incertain embodiments, the degree of homology between the two sequences issufficient to allow homologous recombination therebetween, utilizingnormal cellular mechanisms. Two homologous non-identical sequences canbe any length and their degree of non-homology can be as small as asingle nucleotide (e.g., for correction of a genomic point mutation bytargeted homologous recombination) or as large as 10 or more kilobases(e.g., for insertion of a gene at a predetermined ectopic site in achromosome). Two polynucleotides comprising the homologous non-identicalsequences need not be the same length. For example, an exogenouspolynucleotide (i.e., donor polynucleotide) of between 20 and 10,000nucleotides or nucleotide pairs can be used.

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100. An approximatealignment for nucleic acid sequences is provided by the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics2:482-489 (1981). This algorithm can be applied to amino acid sequencesby using the scoring matrix developed by Dayhoff, Atlas of ProteinSequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, NationalBiomedical Research Foundation, Washington, D.C., USA, and normalized byGribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplaryimplementation of this algorithm to determine percent identity of asequence is provided by the Genetics Computer Group (Madison, Wis.) inthe “BestFit” utility application. The default parameters for thismethod are described in the Wisconsin Sequence Analysis Package ProgramManual, Version 8 (1995) (available from Genetics Computer Group,Madison, Wis.). A preferred method of establishing percent identity inthe context of the present disclosure is to use the MPSRCH package ofprograms copyrighted by the University of Edinburgh, developed by JohnF. Collins and Shane S. Sturrok, and distributed by IntelliGenetics,Inc. (Mountain View, Calif.). From this suite of packages theSmith-Waterman algorithm can be employed where default parameters areused for the scoring table (for example, gap open penalty of 12, gapextension penalty of one, and a gap of six). From the data generated the“Match” value reflects sequence identity. Other suitable programs forcalculating the percent identity or similarity between sequences aregenerally known in the art, for example, another alignment program isBLAST, used with default parameters. For example, BLASTN and BLASTP canbe used using the following default parameters: genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST. Withrespect to sequences described herein, the range of desired degrees ofsequence identity is approximately 80% to 100% and any integer valuetherebetween. Typically the percent identities between sequences are atleast 70-75%, preferably 80-82%, more preferably 85-90%, even morepreferably 92%, still more preferably 95%, and most preferably 98%sequence identity.

Alternatively, the degree of sequence similarity between polynucleotidescan be determined by hybridization of polynucleotides under conditionsthat allow formation of stable duplexes between homologous regions,followed by digestion with single-stranded-specific nuclease(s), andsize determination of the digested fragments. Two nucleic acid, or twopolypeptide sequences are substantially homologous to each other whenthe sequences exhibit at least about 70%-75%, preferably 80%-82%, morepreferably 85%-90%, even more preferably 92%, still more preferably 95%,and most preferably 98% sequence identity over a defined length of themolecules, as determined using the methods above. As used herein,substantially homologous also refers to sequences showing completeidentity to a specified DNA or polypeptide sequence. DNA sequences thatare substantially homologous can be identified in a Southernhybridization experiment under, for example, stringent conditions, asdefined for that particular system. Defining appropriate hybridizationconditions is within the skill of the art. See, e.g., Sambrook et al.,supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D.Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

Selective hybridization of two nucleic acid fragments can be determinedas follows. The degree of sequence identity between two nucleic acidmolecules affects the efficiency and strength of hybridization eventsbetween such molecules. A partially identical nucleic acid sequence willat least partially inhibit the hybridization of a completely identicalsequence to a target molecule. Inhibition of hybridization of thecompletely identical sequence can be assessed using hybridization assaysthat are well known in the art (e.g., Southern (DNA) blot, Northern(RNA) blot, solution hybridization, or the like, see Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.). Such assays can be conducted using varying degreesof selectivity, for example, using conditions varying from low to highstringency. If conditions of low stringency are employed, the absence ofnon-specific binding can be assessed using a secondary probe that lackseven a partial degree of sequence identity (for example, a probe havingless than about 30% sequence identity with the target molecule), suchthat, in the absence of non-specific binding events, the secondary probewill not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a reference nucleic acidsequence, and then by selection of appropriate conditions the probe andthe reference sequence selectively hybridize, or bind, to each other toform a duplex molecule. A nucleic acid molecule that is capable ofhybridizing selectively to a reference sequence under moderatelystringent hybridization conditions typically hybridizes under conditionsthat allow detection of a target nucleic acid sequence of at least about10-14 nucleotides in length having at least approximately 70% sequenceidentity with the sequence of the selected nucleic acid probe. Stringenthybridization conditions typically allow detection of target nucleicacid sequences of at least about 10-14 nucleotides in length having asequence identity of greater than about 90-95% with the sequence of theselected nucleic acid probe. Hybridization conditions useful forprobe/reference sequence hybridization, where the probe and referencesequence have a specific degree of sequence identity, can be determinedas is known in the art (see, for example, Nucleic Acid Hybridization: APractical Approach, editors B. D. Hames and S. J. Higgins, (1985)Oxford; Washington, D.C.; IRL Press).

Conditions for hybridization are well-known to those of skill in theart. Hybridization stringency refers to the degree to whichhybridization conditions disfavor the formation of hybrids containingmismatched nucleotides, with higher stringency correlated with a lowertolerance for mismatched hybrids. Factors that affect the stringency ofhybridization are well-known to those of skill in the art and include,but are not limited to, temperature, pH, ionic strength, andconcentration of organic solvents such as, for example, formamide anddimethylsulfoxide. As is known to those of skill in the art,hybridization stringency is increased by higher temperatures, lowerionic strength and lower solvent concentrations.

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of the sequences, base composition of thevarious sequences, concentrations of salts and other hybridizationsolution components, the presence or absence of blocking agents in thehybridization solutions (e.g., dextran sulfate, and polyethyleneglycol), hybridization reaction temperature and time parameters, as wellas, varying wash conditions. The selection of a particular set ofhybridization conditions is selected following standard methods in theart (see, for example, Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, (1989) Cold Spring Harbor, N.Y.).

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells. This process requires nucleotide sequence homology,uses a “donor” molecule to template repair of a “target” molecule (i.e.,the one that experienced the double-strand break), and is variouslyknown as “non-crossover gene conversion” or “short tract geneconversion,” because it leads to the transfer of genetic informationfrom the donor to the target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or “synthesis-dependent strand annealing,” in which the donor isused to resynthesize genetic information that will become part of thetarget, and/or related processes. Such specialized HR often results inan alteration of the sequence of the target molecule such that part orall of the sequence of the donor polynucleotide is incorporated into thetarget polynucleotide.

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

An “cleavage half-domain” is a polypeptide sequence which, inconjunction with a second polypeptide (either identical or different)forms a complex having cleavage activity (preferably double-strandcleavage activity). The terms “first and second cleavage half-domains;”“+ and − cleavage half-domains” and “right and left cleavagehalf-domains” are used interchangeably to refer to pairs of cleavagehalf-domains that dimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 20050064474 and 20060188987 and U.S.Provisional Application No. 60/808,486 (filed May 25, 2006),incorporated herein by reference in their entireties.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

An “accessible region” is a site in cellular chromatin in which a targetsite present in the nucleic acid can be bound by an exogenous moleculewhich recognizes the target site. Without wishing to be bound by anyparticular theory, it is believed that an accessible region is one thatis not packaged into a nucleosomal structure. The distinct structure ofan accessible region can often be detected by its sensitivity tochemical and enzymatic probes, for example, nucleases.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist. For example, thesequence 5′-GAATTC-3′ is a target site for the Eco RI restrictionendonuclease.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPDNA-binding domain and a cleavage domain) and fusion nucleic acids (forexample, a nucleic acid encoding the fusion protein described supra).Examples of the second type of fusion molecule include, but are notlimited to, a fusion between a triplex-forming nucleic acid and apolypeptide, and a fusion between a minor groove binder and a nucleicacid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of a mRNA. Gene products also include RNAs whichare modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression.

“Eucaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFPDNA-binding domain is fused to a cleavage domain, the ZFP DNA-bindingdomain and the cleavage domain are in operative linkage if, in thefusion polypeptide, the ZFP DNA-binding domain portion is able to bindits target site and/or its binding site, while the cleavage domain isable to cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

Zinc Finger Nucleases

Described herein are zinc finger nucleases (ZFNs) that can be used forgene inactivation, for example inactivation of the CCR5 gene. ZFNscomprise a zinc finger protein (ZFP) and a nuclease (cleavage) domain.

A. Zinc Finger Proteins

Zinc finger binding domains can be engineered to bind to a sequence ofchoice. See, for example, Beerli et al. (2002) Nature Biotechnol.20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan etal. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr.Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct.Biol. 10:411-416. An engineered zinc finger binding domain can have anovel binding specificity, compared to a naturally-occurring zinc fingerprotein. Engineering methods include, but are not limited to, rationaldesign and various types of selection. Rational design includes, forexample, using databases comprising triplet (or quadruplet) nucleotidesequences and individual zinc finger amino acid sequences, in which eachtriplet or quadruplet nucleotide sequence is associated with one or moreamino acid sequences of zinc fingers which bind the particular tripletor quadruplet sequence. See, for example, co-owned U.S. Pat. Nos.6,453,242 and 6,534,261, incorporated by reference herein in theirentireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237.

Enhancement of binding specificity for zinc finger binding domains hasbeen described, for example, in co-owned WO 02/077227.

Selection of target sites; ZFPs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in related to U.S.Publication Nos. 20030232410; 20050208489; 2005064474; 20050026157;20060188987; International Publication WO 07/014,275; U.S. patentapplication Ser. Nos. 10/587,723 (filed Jul. 27, 2006); 11/493,423(filed Jul. 26, 2006), the disclosures of which are incorporated byreference in their entireties for all purposes.

In certain embodiments, the zinc finger nucleases of the Ad-ZFN vectorsdescribed herein bind in a CCR-5 gene. Table 1 describes a number ofzinc finger binding domains that have been engineered to bind tonucleotide sequences in the human CCR-5 gene. Each row describes aseparate zinc finger DNA-binding domain. The DNA target sequence foreach domain is shown in the first column (DNA target sites indicated inuppercase letters; non-contacted nucleotides indicated in lowercase),and the second through fifth columns show the amino acid sequence of therecognition region (amino acids −1 through +6, with respect to the startof the helix) of each of the zinc fingers (F1 through F4) in theprotein. Also provided in the first column is an identification numberfor each protein.

TABLE 1 Zinc finger nucleases targeted to the human CCR-5 geneTarget sequence F1 F2 F3 F4 r162 designs GATGAGGATGAC DRSNLSR TSANLSRRSDNLAR TSANLSR (SEQ ID NO: 1) 7296 (SEQ ID NO: 2) (SEQ ID NO: 3)(SEQ ID NO: 4) (SEQ ID NO: 3) GATGAGGATGAC DRSNLSR ISSNLNS RSDNLARTSANLSR (SEQ ID NO: 1) 8181 (SEQ ID NO: 2) (SEQ ID NO: 5) (SEQ ID NO: 4)(SEQ ID NO: 3) GATGAGGATGAC DRSNLSR VSSNLTS RSDNLAR TSANLSR(SEQ ID NO: 1) 8182 (SEQ ID NO: 2) (SEQ ID NO: 6) (SEQ ID NO: 4)(SEQ ID NO: 3) GATGAGGATGAC DRSNLSR ISSNLNS RSDNLAR NRDNLSR(SEQ ID NO: 1) 8262 (SEQ ID NO: 2) (SEQ ID NO: 5) (SEQ ID NO: 4)(SEQ ID NO: 7) GATGAGGATGAC DRSNLSR ISSNLNS RSDNLAR TSGNLTR(SEQ ID NO: 1) 8266 (SEQ ID NO: 2) (SEQ ID NO: 5) (SEQ ID NO: 4)(SEQ ID NO: 8) GATGAGGATGAC DRSNLSR VSSNLTS RSDNLAR TSGNLTR(SEQ ID NO: 1) 8267 (SEQ ID NO: 2) (SEQ ID NO: 6) (SEQ ID NO: 4)(SEQ ID NO: 8) GATGAGGATGAC DRSNLSR TSGNLTR RSDNLAR TSGNLTR(SEQ ID NO: 1) 7741 (SEQ ID NO: 2) (SEQ ID NO: 8) (SEQ ID NO: 4)(SEQ ID NO: 8) 168 designs AAACTGCAAAAG RSDNLSV QNANRIT RSDVLSE QRNHRTT(SEQ ID NO: 9) 7745 (SEQ ID NO: 10) (SEQ ID NO: 11) (SEQ ID NO: 12)(SEQ ID NO: 13) AAACTGCAAAAG RSDNLSN QNANRIT RSDVLSE QRNHRTT(SEQ ID NO: 9) 8165 (SEQ ID NO: 14) (SEQ ID NO: 11) (SEQ ID NO: 12)(SEQ ID NO: 13) AAACTGCAAAAG RSDNLSV QRVNLIV RSDVLSE QRNHRTT(SEQ ID NO: 9) 8191 (SEQ ID NO: 10) (SEQ ID NO: 15) (SEQ ID NO: 12)(SEQ ID NO: 13) AAACTGCAAAAG RSDNLGV QKINLQV RSDVLSE QRNHRTT(SEQ ID NO: 9) 8196 (SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 12)(SEQ ID NO: 13) AAACTGCAAAAG RSDNLSV QKINLQV RSDVLSE QRNHRTT(SEQ ID NO: 9) 8196z (SEQ ID NO: 10) (SEQ ID NO: 17) (SEQ ID NO: 12)(SEQ ID NO: 13) AAACTGCAAAAG RSDNLGV QKINLQV RSDVLSE QRNHRTT(SEQ ID NO: 9) 8196zg (SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 12)(SEQ ID NO: 13) AAACTGCAAAAG RSDHLSE QNANRIT RSDVLSE QRNHRTT(SEQ ID NO: 9) 7568 (SEQ ID NO: 18) (SEQ ID NO: 11) (SEQ ID NO: 12)(SEQ ID NO: 13) r627 designs GACAAGCAGCGG RSAHLSE RSANLSE RSANLSVDRANLSR (SEQ ID NO: 19) 7524 (SEQ ID NO: 20) (SEQ ID NO: 21)(SEQ ID NO: 22) (SEQ ID NO: 23) 633 designs CATCTGcTACTCG RSDSLSKDNSNRIK RSAVLSE TNSNRIT (SEQ ID NO: 24) 8040 (SEQ ID NO: 25)(SEQ ID NO: 26) (SEQ ID NO: 27) (SEQ ID NO: 28)

As described below, in certain embodiments, a four-finger binding domainas shown in Table 1 is fused to a cleavage half-domain, such as, forexample, the cleavage domain of a Type IIs restriction endonuclease suchas FokI. A pair of such zinc finger/nuclease half-domain fusions areused for targeted cleavage, as disclosed, for example, in U.S. PatentPublication No. 20050064474 (application Ser. No. 10/912,932). Forexample, ZFN-215 denotes the pair of fusion proteins containing the zincfinger binding domains designated 8267 (which recognizes the targetsequence shown in SEQ ID NO:1 and comprises the 4 recognition helicesdepicted in SEQ ID NOs:2, 6, 4 and 8) and 8196z (which recognizes thetarget sequence shown in SEQ ID NO:9 and comprises the 4 recognitionhelices depicted in SEQ ID NOs:10, 17, 12 and 13). ZFN-201 denotes thepair of fusion proteins containing the zinc finger binding domainsdesignated 8266 (which recognizes the target sequence shown in SEQ IDNO:1 and comprises the 4 recognition helices depicted in SEQ ID NOs: 2,2, 4 and 8) and 8196z (which recognizes the target sequence shown in SEQID NO:9 and comprises the 4 recognition helices depicted in SEQ IDNOs:10, 17, 12 and 13).

For targeted cleavage, the near edges of the binding sites can separatedby 5 or more nucleotide pairs, and each of the fusion proteins can bindto an opposite strand of the DNA target. Hence, any one of the proteinsidentified as an “r162 design” in Table 1 (indicating that it binds tothe reverse strand and that the downstream edge of its binding site isat nucleotide 162) can be paired with any of the proteins identified asa “168 design” (indicating that it binds to the strand opposite thatbound by the r162 designs and that the upstream edge of its binding siteis at nucleotide 168). For example, protein 8267 can be paired withprotein 8196 or with protein 8196z or with any of the other 168 designs;and protein 8266 can be paired with either of proteins 8196 or 8196z orwith any other of the 168 designs. All pairwise combinations of the r162and 168 designs can be used for targeted cleavage and mutagenesis of aCCR-5 gene. Similarly, the 7524 protein (or any other r627 design) canbe used in conjunction with the 8040 protein (or any other 633 design)to obtain targeted cleavage and mutagenesis of a CCR-5 gene.

The CCR5-ZFNs described herein can be targeted to any sequence in theCCR5 genome. For example, CCR5 genomic sequences (including allelicvariants such as CCR5-Δ32) are well known in the art and individualshomozygous for the CCR5-Δ32 (see, e.g., Liu et al. (1996) Cell 367-377),are resistant to HIV-1 infection.

B. Cleavage Domains

The ZFNs also comprise a nuclease (cleavage domain, cleavagehalf-domain). The cleavage domain portion of the fusion proteinsdisclosed herein can be obtained from any endonuclease or exonuclease.Exemplary endonucleases from which a cleavage domain can be derivedinclude, but are not limited to, restriction endonucleases and homingendonucleases. See, for example, 2002-2003 Catalogue, New EnglandBiolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res.25:3379-3388. Additional enzymes which cleave DNA are known (e.g., S1Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease;yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, ColdSpring Harbor Laboratory Press, 1993). One or more of these enzymes (orfunctional fragments thereof) can be used as a source of cleavagedomains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme FokI catalyzes double-strandedcleavage of DNA, at 9 nucleotides from its recognition site on onestrand and 13 nucleotides from its recognition site on the other. See,for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as wellas Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al.(1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc.Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise thecleavage domain (or cleavage half-domain) from at least one Type IISrestriction enzyme and one or more zinc finger binding domains, whichmay or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the Fok I enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-Fok Ifusions are provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014,275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 20050064474 and 20060188987(application Ser. Nos. 10/912,932 and 11/304,981, respectively) and inU.S. provisional patent application No. 60/808,486 (filed May 25, 2006),the disclosures of all of which are incorporated by reference in theirentireties herein. Amino acid residues at positions 446, 447, 479, 483,484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 ofFok I are all targets for influencing dimerization of the FokI cleavagehalf-domains.

Exemplary engineered cleavage half-domains of FokI that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499. See FIGS. 2, 3 and 4.

Thus, in one embodiment, as shown in FIGS. 3 and 4, the mutation at 490replaces Glu (E) with Lys (K); the mutation at 538 replaces Iso (I) withLys (K); the mutation at 486 replaced Gln (Q) with Glu (E); and themutation at position 499 replaces Iso (I) with Lys (K). Specifically,the engineered cleavage half-domains described herein were prepared bymutating positions 490 (E→K) and 538 (I→K) in one cleavage half-domainto produce an engineered cleavage half-domain designated “E490K:I538K”and by mutating positions 486 (Q→E) and 499 (I→L) in another cleavagehalf-domain to produce an engineered cleavage half-domain designated“Q486E:I499L”. The engineered cleavage half-domains described herein areobligate heterodimer mutants in which aberrant cleavage is minimized orabolished. See, e.g., Example 1 of U.S. Provisional Application No.60/808,486 (filed May 25, 2006), the disclosure of which is incorporatedby reference in its entirety for all purposes.

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. PatentPublication No. 20050064474 (Ser. No. 10/912,932, Example 5) and U.S.Patent Provisional Application Ser. No. 60/721,054 (Example 38).

C. Additional Methods for Targeted Cleavage in CCR5

Any nuclease having a target site in a CCR5 gene can be used in themethods disclosed herein. For example, homing endonucleases andmeganucleases have very long recognition sequences, some of which arelikely to be present, on a statistical basis, once in a human-sizedgenome. Any such nuclease having a unique target site in a CCR5 gene canbe used instead of, or in addition to, a zinc finger nuclease, fortargeted cleavage in a CCR5 gene.

Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce,I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI,I-TevII and I-TevIII. Their recognition sequences are known. See alsoU.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort et al. (1997)Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118;Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996)Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol.263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the NewEngland Biolabs catalogue.

Although the cleavage specificity of most homing endonucleases is notabsolute with respect to their recognition sites, the sites are ofsufficient length that a single cleavage event per mammalian-sizedgenome can be obtained by expressing a homing endonuclease in a cellcontaining a single copy of its recognition site. It has also beenreported that the specificity of homing endonucleases and meganucleasescan be engineered to bind non-natural target sites. See, for example,Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003)Nucleic Acids Res. 31:2952-2962; Ashworth et al. (2006) Nature441:656-659; Paques et al. (2007) Current Gene Therapy 7:49-66.

Delivery

The ZFNs described herein may be delivered to a target cell by anysuitable means. Methods of delivering proteins comprising zinc fingersare described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717;6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113;6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which areincorporated by reference herein in their entireties.

ZFNs as described herein may also be delivered using vectors containingsequences encoding one or more ZFNs. Any vector systems may be usedincluding, but not limited to, plasmid vectors, retroviral vectors,lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirusvectors and adeno-associated virus vectors, etc. See, also, U.S. Pat.Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219;and 7,163,824, incorporated by reference herein in their entireties.

In certain embodiments, the vector is an adenovirus vector. Thus,described herein are adenovirus (Ad) vectors for introducingheterologous sequences (e.g., zinc finger nucleases (ZFNs)) into cells.

Non-limiting examples of Ad vectors that can be used in the presentapplication include recombinant (such as E1-deleted), conditionallyreplication competent (such as oncolytic) and/or replication competentAd vectors derived from human or non-human serotypes (e.g., Ad5, Ad11,Ad35, or porcine adenovirus-3); and/or chimeric Ad vectors (such asAd5/35) or tropism-altered Ad vectors with engineered fiber (e.g., knobor shaft) proteins (such as peptide insertions within the HI loop of theknob protein). Also useful are “gutless” Ad vectors, e.g., an Ad vectorin which all adenovirus genes have been removed, to reduceimmunogenicity and to increase the size of the DNA payload. This allows,for example, simultaneous delivery of sequences encoding ZFNs and adonor sequence. Such gutless vectors are especially useful when thedonor sequences include large transgenes to be integrated via targetedintegration.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer, and they readily infect a number of differentcell types. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in cells that provide one or more of thedeleted gene functions in trans. For example, human 293 cells supply E1function. Ad vectors can transduce multiple types of tissues in vivo,including non-dividing, differentiated cells such as those found inliver, kidney and muscle. Conventional Ad vectors have a large carryingcapacity. An example of the use of an Ad vector in a clinical trialinvolved polynucleotide therapy for antitumor immunization withintramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-1089(1998)).

Additional examples of the use of adenovirus vectors for gene transferin clinical trials include Rosenecker et al., Infection 24:1 5-10(1996); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarez et al.,Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther. 5:507-513(1998).

In certain embodiments, the Ad vector is a chimeric adenovirus vector,containing sequences from two or more different adenovirus genomes. Forexample, the Ad vector can be an Ad5/35 vector. Ad5/35 is created byreplacing one or more of the fiber protein genes (knob, shaft, tail,penton) of Ad5 with the corresponding fiber protein gene from a B groupadenovirus such as, for example, Ad35. The Ad5/35 vector andcharacteristics of this vector are described, for example, in Ni et al.(2005) “Evaluation of biodistribution and safety of adenovirus vectorscontaining group B fibers after intravenous injection into baboons,” HumGene Ther 16:664-677; Nilsson et al. (2004) “Functionally distinctsubpopulations of cord blood CD34+ cells are transduced by adenoviralvectors with serotype 5 or 35 tropism,” Mol Ther 9:377-388; Nilsson etal. (2004) “Development of an adenoviral vector system with adenovirusserotype 35 tropism; efficient transient gene transfer into primarymalignant hematopoietic cells,” J Gene Med 6:631-641; Schroers et al.(2004) “Gene transfer into human T lymphocytes and natural killer cellsby Ad5/F35 chimeric adenoviral vectors,” Exp Hematol 32:536-546;Seshidhar et al. (2003) “Development of adenovirus serotype 35 as a genetransfer vector,” Virology 311:384-393; Shayakhmetov et al. (2000)“Efficient gene transfer into human CD34(+) cells by a retargetedadenovirus vector,” J Virol 74:2567-2583; and Sova et al. (2004), “Atumor-targeted and conditionally replicating oncolytic adenovirus vectorexpressing TRAIL for treatment of liver metastases,” Mol Ther 9:496-509.

As noted above, ZFNs and polynucleotides encoding these ZFNs may bedelivered to any target cell. Generally, for inactivating a gene CCR-5,the cell is an immune cell, for example, a lymphocyte (B-cells, T-cellssuch as T helper (T_(H)) and T cytotoxic cells (T_(C)), null cells suchas natural killer (NK) cells); a mononuclear cell (monocytes,marcophages); a granulocytic cell (granulocytes, neutrophils,eosinophils, basophils); a mast cell; and/or a dendritic cell(Langerhans cells, interstitial dendritic cells, interdigitatingdendritic cells, circulating dendritic cells). Macrophages, Blymphocytes and dendritic cells are exemplary antigen-presenting cellsinvolved in T_(H) cell activation. In certain embodiments, the targetcell is a T_(H) cell, characterized by expression of CD4 on the surface.The target cell may also be a hematopoietic stem cell, which may giverise to any immune cell.

Applications

The disclosed methods and compositions can be used to cleave DNA at aregion of interest in cellular chromatin (e.g., at a desired orpredetermined site in a genome, for example, in a gene, either mutant orwild-type); to replace a genomic sequence (e.g., a region of interest incellular chromatin, see, also, Example 5 below) with a homologousnon-identical sequence (i.e., targeted recombination); to delete agenomic sequence by cleaving DNA at one or more sites in the genome,which cleavage sites are then joined by non-homologous end joining(NHEJ); to screen for cellular factors that facilitate homologousrecombination; to replace a wild-type sequence with a mutant sequence;and/or to convert one allele to a different allele. Such methods alsoallow for generation and/or modification of cells lines (for therapeuticand non-therapeutic uses), treatment of infections (viral or bacterial)in a host (e.g., by blocking expression of viral or bacterial receptors,thereby preventing infection and/or spread in a host organism); to treatgenetic diseases.

Thus, the compositions and methods described herein can be used for genemodification, gene correction, and gene disruption. Non-limitingexamples of gene modification includes homology directed repair(HDR)-based targeted integration; HDR-based gene correction; HDR-basedgene modification; HDR-based gene disruption; NHEJ-based gene disruptionand/or combinations of HDR, NHEJ, and/or single strand annealing (SSA).Single-Strand Annealing (SSA) refers to the repair of a double strandbreak between two repeated sequences that occur in the same orientationby resection of the DSB by 5′-3′ exonucleases to expose the 2complementary regions. The single-strands encoding the 2 direct repeatsthen anneal to each other, and the annealed intermediate can beprocessed such that the single-stranded tails (the portion of thesingle-stranded DNA that is not annealed to any sequence) are bedigested away, the gaps filled in by DNA Polymerase, and the DNA endsrejoined. This results in the deletion of sequences located between thedirect repeats.

The compositions (e.g., Ad-ZFN vectors) and methods described herein canalso be used in the treatment of various genetic diseases and/orinfectious diseases.

The compositions and methods can also be applied to stem cell basedtherapies, including but not limited to:

(a) Correction of somatic cell mutations by short patch gene conversionor targeted integration for monogenic gene therapy

(b) Disruption of dominant negative alleles

(c) Disruption of genes required for the entry or productive infectionof pathogens into cells

(d) Enhanced tissue engineering, for example, by:

(i) Modifying gene activity to promote the differentiation or formationof functional tissues; and/or

(ii) Disrupting gene activity to promote the differentiation orformation of functional tissues

(e) Blocking or inducing differentiation, for example, by:

(i) Disrupting genes that block differentiation to promote stem cells todifferentiate down a specific lineage pathway

(ii) Targeted insertion of a gene or siRNA expression cassette that canstimulate stem cell differentiation.

(iii) Targeted insertion of a gene or siRNA expression cassette that canblock stem cell differentiation and allow better expansion andmaintenance of pluripotency

(iv) Targeted insertion of a reporter gene in frame with an endogenousgene that is a marker of pluripotency or differentiation state thatwould allow an easy marker to score differentiation state of stem cellsand how changes in media, cytokines, growth conditions, expression ofgenes, expression of siRNA molecules, exposure to antibodies to cellsurface markers, or drugs alter this state.

(f) Somatic cell nuclear transfer, for example, a patient's own somaticcells can be isolated, the intended target gene modified in theappropriate manner, cell clones generated (and quality controlled toensure genome safety), and the nuclei from these cells isolated andtransferred into unfertilized eggs to generate patient-specific hEScells that could be directly injected or differentiated beforeengrafting into the patient, thereby reducing or eliminating tissuerejection.

(g) Universal stem cells by knocking out MHC receptors—This approachwould be used to generate cells of diminished or altogether abolishedimmunological identity. Cell types for this procedure include but arenot limited to, T-cells, B cells, hematopoietic stem cells, andembryonic stem cells. Therefore, these stem cells or their derivatives(differentiated cell types or tissues) could be potentially engraftedinto any person regardless of their origin or histocompatibility.

The compositions and methods can also be used for somatic cell therapy(e.g., autologous cell therapy and/or universal T-cell by knocking outMHC receptors, see section (g) above), thereby allowing production ofstocks of T-cells that have been modified to enhance their biologicalproperties. Such cells can be infused into a variety of patientsindependent of the donor source of the T-cells and theirhistocompatibility to the recipient.

In addition, the use of Ad vectors as described herein to deliver ZFNsenhances rates of NHEJ. Without being bound by one theory, it appearsthat this effect is due to the inhibitory effect the E4 proteins (E4ORF6 (E4 34k), E4 ORF3) may have on DSB repair.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entireties.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity and understanding,it will be apparent to those of skill in the art that various changesand modifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing disclosure andfollowing examples should not be construed as limiting.

EXAMPLES Example 1 Vector Construction

A. Construction of Ad5/35-GFP and Ad5/35-ZFN Vectors

Chimeric Ad5/35 vectors (Nilsson et al. (2004) Mol Ther 9:377-388;Nilsson et al. (2004) J Gene Med 6:631-641) were constructed as shownschematically in FIG. 1. Briefly, a sequence encoding an expressioncassette for GFP (top line), an expression cassette for a pair ofengineered ZFNs (ZFN1 and ZFN2) which target the endogenous CCR5 locus(middle line), or a homologous donor sequence for targeting a 47-bppatch sequence in the CCR5 locus (bottom line) was inserted in place ofthe E1 genes using the AdEasy bacterial recombination system(Stratagene).

The Ad-215 and Ad-201 vector constructs each comprise sequences encodingtwo ZFNs that cleave the endogenous CCR5 gene at sequences encodingLeu55. Both ZFNs are expressed under the control of a single promotervia a 2A fusion. As shown in Table 1 above, the ZFN215 pair encoded bythe Ad-215 construct comprise zinc finger binding domains 8267 and 8196zfrom the r162 and 168 designs, respectively; while the ZFN201 pairencoded by the Ad-201 vector comprises zinc finger binding domains 8266and 8196z from the r162 and 168 designs, respectively. The ZFNs encodedby the Ad-215 and Ad-201 vectors are fused to a wild-type FokI cleavagehalf-domain. See FIG. 2.

The ZFN224 pair encoded by the Ad-224 construct comprise zinc fingerbinding domains 8267 and 8196z from the r162 and 168 designs,respectively. Thus, The Ad-224 construct has the same zinc fingerproteins as Ad-215. However, the ZFNs encoded by the Ad-224 constructcontain mutant FokI cleavage-half domain as described in U.S.Provisional Application No. 60/808,486 (filed May 25, 2006), andincorporated herein by reference in its entirety. In particular, the8267 zinc finger domain is fused to a FokI cleavage half-domaincontaining the Q486E and I499L mutations (FIG. 3), and 8196z zinc fingerdomain is fused to a FokI cleavage half-domain containing the E490K andI538K mutations (FIG. 4).

B. Donor Vector for Targeted Insertion

To make a donor vector (Ad5/35 P ori, FIG. 1 bottom line), an 1881-bpfragment of the human genome corresponding to the CCR5 locus was PCRamplified and cloned into the PCR4-TOPO vector (Invitrogen). Thesequence of the fragment is shown in FIG. 5 (SEQ ID NO:36).

In order to generate a cloning site at which to insert a “patch”sequence, 2 nucleotides of the sequence shown in FIG. 5 were changed togenerate a XbaI recognition site. Specifically, the nucleotide sequence“atcctgataa” (SEQ ID NO:39) (nucleotides 470-479 in the donor fragment)was changed to “atctagataa” (SEQ ID NO:40) (the 2 bases changed areunderlined) via the QuickChange Site-Directed Mutagenesis Kit(Stratagene).

The resulting DNA sequence was then digested with XbaI, and a 47 bp“patch” sequence, shown in FIG. 6 (SEQ ID NO:37), was inserted at theXbaI site.

The resulting sequence, shown in FIG. 7 (SEQ ID NO:38), was insertedinto the Ad5/35 vector as described above for the GFP and ZFN expressioncassettes. With respect to the sequence shown in FIG. 7, the 5′homologyarm correspond to nucleotides 1-471; the “patch” sequence for targetedinsertion into CCR-5 is underlined and corresponds to nucleotides472-518; and the 3′ homology arm corresponds to nucleotides 519-1928.

Example 2 Transduction of hES Cells with Ad5/35-GFP Vectors

Chimeric Ad5/35-GFP vectors as described in Example 1 were introducedinto human embryonic stem (hES) cells as follows.

Infections of hES cells were performed in 500 μl volumes using 400,000cells and 25 μl, 5 μl or 0.5 μl of the Ad5/35-GFP vector (MOI of 8200,1640 and 164 respectively). After 4 hours, the cells were washed andplated onto fresh murine embryonic fibroblast (MEF) feeder cells.Fluorescence microscopy of living cells, obtained approximately 20 hourspost-infection, showed fluorescence in stem cell colonies that had beeninfected with 5 and 25 μl of virus, and no fluorescence in the feedercells. FACS analysis for GFP fluorescence was performed ˜22 hourspost-infection. The results are shown in Table 2.

TABLE 2 Infection % of fluorescent T-cells Mock infection 0.74%Ad5/35-GFP 0.5 μl 39.4% Ad5/35-GFP 5 μl 91% Ad5/35-GFP 25 μl 95%

These results indicate that Ad5/35 vectors are capable of infectinghuman embryonic stem cells at high efficiency.

Example 3 Modification of the CCR-5 Gene Using Ad5/35-ZFNs

CD4⁺ T-cells and PBMCs were obtained from AllCells. Cells were culturedin RPMI+10% FBS+1% L-Glutamine (30 mg/mL)+IL-2 (1 ng/mL, Sigma) andactivated with anti CD3-CD28 beads according to manufacturer's protocol(Dynal). Cells were seeded at 3E5 cell/mL in 1 mL volume in a 24 wellplate.

Adenoviral vectors as described in Example 1 (Ad5/35 GFP, Ad5/35 215 orAd5/35 224) were added two days later at an MOI of 10, 30, or 100 (MOIcalculated based on infectious titer).

Cells were harvested 2 days after exposure to virus and genemodification efficiency was determined using a Cel-1 assay, performed asdescribed in International Patent Publication WO 07/014,275. See, also,Oleykowski et al. (1998) Nucleic Acids res. 26:4597-4602; Qui et al.(2004) BioTechniques 36:702-707; Yeung et al. (2005) BioTechniques38:749-758.

Results are shown in Table 3.

TABLE 3 % CCR-5 Alleles Modified PBMCs CD4+ T-cells Control notdetectable not detectable Ad5/35-ZFN215 at MOI 10 6.1 6.7 Ad5/35-ZFN215at MOI 30 14.0 16.5 Ad5/35-ZFN215 at MOI 100 31.2 32.3 Ad5/35-ZFN224 atMOI 10 3.6 1.8 Ad5/35-ZFN224 at MOI 30 7.4 9.0 Ad5/35-ZFN224 at MOI 10015.0 14.9 Ad5/35-GFP at MOI 10 not detectable not detectable Ad5/35-GFPat MOI 30 not detectable not detectable

These results indicate that gene modification was observed afterinfection of cells with Ad5/35 vectors encoding both the ZFN215 nucleasepair (comprising wild type FokI cleavage half-domains) and the ZFN 224nuclease pair (comprising the mutant FokI cleavage half-domainsdescribed in Example 1A). In addition, the results show that genemodification levels increased in a dose-dependent manner.

To determine the persistence of ZFN-induced gene modification, infectedcells were kept in culture for another 8 days (same culture medium asbefore). Cells were counted and diluted with fresh medium every 2 days.Cells were then harvested (10 days after viral transduction) and theCel-1 assay was repeated. In addition, a fraction of the CD4⁺ T-cellpopulation was re-activated with anti CD3/CD28 beads (Dynal) on day 7.Results are shown in Table 4.

TABLE 4 % Cells Infected Re-activated CD4 Non-activated CD4 Control notdetectable not detectable Ad5/35-ZFN215 at MOI 30 10.1 10.9Ad5/35-ZFN215 at MOI 100 29.0 28.2 Ad5/35-ZFN224 at MOI 30 6.7 6.4Ad5/35-ZFN224 at MOI 100 16.1 16.6 Ad5/35-GFP at MOI 100 not detectablenot detectable

These results show that the degree of gene modification was maintainedas the CD4⁺ T-cells expanded. Furthermore, reactivation had no apparenteffect on the growth of the modified cells as compared to unmodifiedcells.

Example 4 Modification of the CCR-5 Gene Using Ad5/35-ZFNs in CD34+Cells

CD4⁺ T-cells and CD34⁺ cells were obtained from AllCells. On day 0, CD4⁺T-cells were cultured in RPMI+10% FBS+1% L-Glutamine (30 mg/mL)+IL-2 (1ng/mL, Sigma) and activated with anti CD3-CD28 beads according to themanufacturer's protocol (Dynal). CD34⁺ cells were cultured in serum freemedium (Stemspan H3000, Stem Cell Technologies) and supplemented withcytokines (Stemspan CC100, Stem Cell Technologies). Cells were seeded at6E5 cell/mL in 1 mL volume in 24 well plates. Adenoviral vectors (Ad5/35GFP or Ad5/35 224) were added the next day (day 1) at different MOIs(MOI calculated based on infectious titer).

Cells were harvested on day 4 and gene modification efficiencies weredetermined using a Cel-1 assay.

For CD4⁺ T-cells, Ad5/35 224 induced CCR-5 gene modification with anefficiency of 18.0%, 34.5% and 48.4% at MOIs of 25, 50 and 100,respectively.

Similarly, for CD34⁺ cells, Ad5/35 224 induced CCR5 gene modification atefficiencies of 10.9% and 11.1%, at MOIs of 10 and 50, respectively.

Example 5 Targeted Insertion of an Exogenous Sequence into the CCR-5Gene Using Ad5/35-ZFNs

On day 0, CD4⁺ T-cells were cultured in RPMI+10% FBS+1% L-Glutamine (30mg/mL)+IL-2 (1 ng/mL, Sigma) and activated with anti CD3-CD28 beadsaccording to the manufacturer's protocol (Dynal). Cells were seeded at3E5 cells/mL in 1 mL volume in a 24 well plate. Two days later (day 2),cells were co-transduced with different combinations of Ad5/35 224 andAd5/35 P on donor (FIGS. 1 and 7). Ad5/35 224 was added at MOIs of 0,25, 50, and 100, and Ad5/35 P ori donor was added at MOIs of 0, 100, and300 (MOI calculated based on infectious titer). Cells were harvested 2days after infection (day 4) and the targeted integration efficiency wasdetermined by RFLP assay, as follows.

Genomic DNA was isolated from transduced cells and PCR amplified withprimers outside the region of donor homology. The amplified fragment wasthen incubated with the restriction enzyme BglI, whose recognition siteis contained within the P ori donor (Patch) sequence. The frequency oftargeted integration of the Patch sequence was calculated by determiningthe ratio of cleaved to un-cleaved products. The frequency of targetedintegration of the Patch sequence was 3.1% when cells were co-transducedwith Ad5/35 224 at an MOI of 50 and Ad5/35 P ori at an MOI of 300.

Example 6 Non-Homologous End Joining Induced Using Ad5/35-ZFPs

To determine the types of ZFN-mediated mutations generated by targetedcleavage followed by non-homologous end joining (NHEJ), genomic CCR-5sequence of modified cells were sequenced and analyzed. Briefly, PBMCsand CD4⁺ T-cells, transduced with Ad5/35 ZFN 215 in the same manner asdescribed in Example 3, were harvested and genomic DNA was extractedfrom these cells. The CCR5 locus was then PCR amplified, Topo clonedinto the PCR4-TOPO vector (Invitrogen), and bacterial clones weresequence analyzed and compared to the wild-type CCR5 locus.

Mutations induced by targeted ZFN cleavage included both deletions andinsertions, and the size of such changes varied over a wide range. Bothdeletions and insertions as short as a single nucleotide pair wereobserved, as were insertions of up to almost 100 nucleotide pairs. Thesequences of some exemplary mutations induced by targeted, ZFN-mediatedcleavage are shown in FIGS. 8-10.

FIG. 8 shows the sequences of a number of deletions identified duringthe analysis. Missing base pairs are denoted with periods.

An insertion of 5 base pairs occurred at a high frequency in ZFN-treatedcells. This 5 base-pair insertion is a duplication of the 5 base-pairsequence between the ZFN-binding sites, converting the sequenceGTCATCCTCATCCTGATAAACTGCAAAAG (SEQ ID NO:41) toGTCATCCTCATCCTGATCTGATAAACTGCAAAAG (SEQ ID NO:42), with the insertedsequence underlined.

Another frequently-observed mutation was a four base-pair insertionbetween the two ZFN binding sites, converting the sequenceGTCATCCTCATCCTGATAAACTGCAAAAG (SEQ ID NO:41) toGTCATCCTCATCCTTCTAGATAAACTGCAAAAG (SEQ ID NO:45), with the insertedsequence underlined.

FIG. 9 shows the nucleotide sequences of additional mutations. In onecase, a combination of a one-nucleotide deletion and a five-nucleotideinsertion was observed.

FIG. 10 shows the sequences of a number of longer insertions resultingfrom targeted ZFN cleavage.

A summary of the sequence modifications to the CCR-5 locus is shownbelow in Table 5.

TABLE 5 NUMBER IN EACH CELL TYPE TYPE OF MUTATION PBMCs CD4+ T-cellsTOTALS Deletion 10 8 18 Insertions 5 bp 11 14 25 4 bp 8 2 10 Other 3 3 6long 1 3 4 Wild-type 10 14 24 Totals 43 44 87

Example 7 Cell Viability Post Ad5/35-ZFN Transduction

Cell viability post-transduction with Ad5/35-ZFN constructs was alsoassessed. Briefly, CD4⁺ T-cells and PBMCs were obtained from AllCells.On day 0, cells were cultured in RPMI+10% FBS+1% L-Glutamine (30mg/mL)+IL-2 (1 ng/mL, Sigma) and activated with anti CD3-CD28 beadsaccording to the manufacturer's protocol (Dynal). Cells were seeded at3E5 cell/mL in 1 mL volume in 24 well plates. Adenoviral vectors (Ad5/35GFP, Ad5/35 215 or Ad5/35 224) were added two days later (day 2) at MOIof 10, 30, and 100 (MOI calculated based on infectious titer). Cellcounts and cell viability were measured at days 4, 6, 8, 10 and 12 usingthe VIACOUNT protocol provided with the GUAVA analytic flow cytometer,following the manufacturer's instructions (Guava Technologies).

Ad5/35 ZFNs were generally well tolerated, with at least 75% of thecells (up to 90% at the lower MOI) being viable at all time points.Thus, minimal toxicity was observed.

Example 8 Cell Growth Post Ad5/35-ZFN Transduction

Cell growth (doubling) post-transduction with Ad5/35-ZFN constructs wasalso assessed.

On day 0, CD4+ T-cells and PBMCs were cultured in RPMI+10% FBS+1%L-Glutamine (30 mg/mL)+IL-2 (1 ng/mL, Sigma). Cells were activated ondays 0 and 6 with anti CD3-CD28 beads according to the manufacturer'sprotocol (Dynal). Cells were initially seeded at 3E5 cell/mL in 1 mLvolume in 24 well plates. Adenoviral vectors (Ad5/35 GFP, Ad5/35 215 orAd5/35 224) were added on day 2 at MOIs of 10, 30, or 100 (MOIcalculated based on infectious titer). Cell counts and cell viabilitywere measured using the VIACOUNT protocol provided with the GUAVAanalytic flow cytometer as described in Example 7.

Cell growth or doubling was minimally affected by the adenovirus (exceptfor Ad5/35 215 at a MOI of 100). Overall, at least 8 doublings(i.e., >100-fold expansion) were achieved over a 14 day period in bothCD4⁺ T-cells and PBMCs.

Example 9 Measuring Persistence of Adenovirus Genome in CD4⁺ T-Cells

CD4+ T-cells were cultured in RPMI+10% FBS+1% L-Glutamine (30mg/mL)+IL-2 (1 ng/mL, Sigma) and activated with anti CD3-CD28 beadsaccording to the manufacturer's protocol (Dynal). Cells were seeded at6E5 cell/mL in 1 mL volume in a 24 well plate. Adenoviral vectors(Ad5/35 215 or Ad5/35 224) were added the next day at MOI of 10, 30 and100 (MOI calculated based on infectious titer). Cells were harvested ondays 4 and 14. DNA was extracted with a Masterpure kit (EpicenterBiotechnologies). Persistence of adenoviral genomes was quantified bypresence of Ad genomic DNA as measured by TaqMan® PCR (AppliedBiosystem). Primer/probes were designed to target and detect the E4region of the adenoviral genome. Detection limit of the TaqMan® PCRprotocol is ˜10⁻⁴ adenoviral genome per cell.

Overall, between 2 days and 12 days post-transduction, the level ofadenoviral genomes per cell decreased by 100-1000 fold. Less than 10⁻²genome per cell was detected at the highest MOI (100) by day 12 posttransduction.

Example 10 Measuring Persistence of Protein Expression in CD4⁺ T-Cells

On day 0, CD4+ T-cells were cultured and activated as described inExample 9. Adenoviral vectors (Ad5/35 GFP) were added the day afteractivation (day 1) at MOIs of 50, 100, 250 and 500 (MOI calculated basedon infectious titer). Percent GFP-positive cells and mean fluorescentintensity (MFI) was determined by GUAVA analytical flow cytometry every2-3 days.

Significant GFP expression was observed initially (day 3) at all MOIs.GFP fluorescence persisted through day 13, from 80-100% of cells GFPpositive on day 3 to 30-60% of cells GFP positive on day 13. However,MFI decreased significantly over the same period (by almost 100 fold),suggesting that even though cells on day 13 were scored as GFP positiveby the flow cytometer, they contained significantly less GFP protein. Inaddition, it is well known that GFP has a relatively long half-life (>24hrs).

Example 11 Measuring ZFN mRNA in CD4⁺ T-Cells Transduced with Ad5/35-ZFNVectors

On day 0, CD4+ T-cells were cultured and activated as described inExample 9. Adenoviral vectors (Ad5/35 215 or Ad5/35 224) were added thenext day (day 1) at MOIs of 30 and 100 (MOI calculated based oninfectious titer). Cells were harvested on days 3 and 9 (i.e., 2 and 8days post-transduction), and RNA was extracted with a High Pure® RNAisolation kit (Roche). ZFN mRNA was quantified by RT-TaqMan® PCR(Applied Biosystems). The primer/probe set was designed and optimized toanneal to sequences encoding the Fok I cleavage half-domain.

Significant amounts of ZFN mRNA were detected (1×10⁵-1×10⁶ copies percell depending on MOI) 2 days post-transduction. However, by 8 dayspost-transduction, ZFN mRNA levels in all but one sample (Ad5/35 215 atMOI of 100) were below the detection limit of the assay. Approximately100 copies/cell were detected in the Ad5/35 215 MOI 100 sample;representing a thousand-fold decrease in mRNA levels between 2 and 8days post-transduction.

Example 12 Disruption of the CCR-5 Gene in Primary Human CD4⁺ T-CellsUsing Ad5/35-ZFN Vectors

Primary human CD4⁺ T-cells (obtained from donors at Univ. ofPennsylvania) were mock transduced or transduced with either the Ad5/35GFP, Ad5/35 215 or Ad5/35 224.

On day 1, the T-cells were pelleted and resuspended to a concentrationof 1×10⁶/ml in Xvivo 15 (BioWhittaker, Walkersville, Md.) supplementedwith 10% fetal calf serum and 1% L-Glutamine. One ml of cells wasexposed to anti-CD3/CD28 beads (prepared as described by Levine et al.(1997) J. Immunol. 159:5921-5930). The next day (day 2) 1×10⁶ cells weretransduced with the Ad5/35 GFP, Ad5/35 215 or Ad5/35 224 vectors at anMOI of 30 or 100. The following day the volume of medium was doubledwith Xvivol5 containing 10% fetal calf serum, 1% L-Glutamine, 0.9%N-acetylcysteine, and 300 IU/ml human recombinant IL-2. For eachcondition, cells were counted every 2 days on a Coulter Counter, a testsample was pelleted for analysis, and the remaining culture was seededand fed to 1×10⁶ cells/ml. On day 6, beads were removed using a magnet,and the cells were cultured with the Xvivol5/FCS/L-Glut/NAC/IL2 mediumdescribed above. On-day 8, a fraction of cells from each sample waspelleted for analysis by Cel-1 and the remaining cells in each sample(at a concentration of 1×10⁶ cells/ml) were infected with the CCR5tropic HIV-1 strain US1 at an MOI of 0.1 (see Example 13).

Every two days cells were counted, pelleted, a small amount wascollected for analysis by Cel-1, and the remaining cells were seeded andfed with the same Xvivo15/FCS/L-glut/NAC/IL2 containing medium.

On Day 13, re-stimulation of CD4+ T-cell cultures was performed with amix of irradiated (3000 rad) allogeneic PBMCs and irradiated (10,000rad) K562 cells expressing CD32 plus OKT3 (anti-CD3) and anti-CD28.

Genomic DNA was harvested 13 days post-transduction and the CCR5disruption efficiency was measured by the Cel-1 assay. Results are shownin Table 6.

TABLE 6 Ad/ZFN Ad/ZFN GFP 215 224 MOI Control 30 100 30 100 30 100Percentage of alleles 0 0 0 54 44 44 30 modified

Example 13 HIV Challenge

In addition, the primary human CD4⁺ T-cells transduced with Ad5/35vectors as described in Example 12 were diluted 1:3 with untransducedcells and either mock-infected or infected (MOI of 0.1) with areplication competent HIV strain, US1. These cells were then passaged toallow multiple rounds of HIV infection and replication. A small amountof cells were isolated on each of days 0, 5, 11, and 17 post-infectionfrom both the mock- and HIV-infected cultures to monitor the percentageof cells containing disrupted CCR5 genes. Genomic DNA was isolated fromeach cell sample and the presence of mutant CCR5 alleles was determinedusing a Cel-1 assay.

Results are shown in FIG. 11 panels A (Ad5/35 215 transduced cells) andB (Ad5/35 224 transduced cells) and indicate that, in cells that hadbeen transfected with Ad ZFN vectors, the number of cells containingsequence alterations in their CCR-5 gene increased after HIV infection(ZFN HIV) but did not increase in cells that had not been infected withHIV (ZFN mock).

Example 14 ZFN Disruption of CCR5 in GHOST-CCR5 Cells

A. Determination of ZFN-Induced Mutations in GHOST-CCR5 Cells

GHOST-CCR5 cells, a reporter cell line for HIV-1 infection containingmultiple (˜4) copies of an autologous CCR5 expression cassette and aninducible GFP marker gene under the control of the HIV-2 LTR (Morner etal. (1999) J. Virol. 73:2343-2349), were obtained from the NIH AIDSResearch and Reference Reagent Program and transduced with an Adenovirus(Ad5/35) vector (Schroers et al. (2004) Experimental Hematology32:536-546) encoding the CCR5-ZFN pairs 215 (see above text related toTable 1) and 224 (containing the ZFNs denoted in Table 1 as 8196z and8266). Binding sites for both of these of the ZFN pairs are the same andare shown in FIG. 12.

Induction of ZFN-mediated mutations at the target site was determinedusing an assay based upon the Surveyor™ nuclease (Transgenomic), alsoknown as Cel-1, a mismatch sensitive enzyme that cleaves DNA at the siteof ZFN-induced mutations. Briefly, genomic DNA was extracted frommodified and control cells using the MasturePure™ DNA purification kit(Epicentre Biotechnologies) and supplemented with 5 uCi α-P³² dATP and 5uCi α-P³² dCTP for radioactive PCR.

Radioactive PCR (50 μl reactions) was performed (AccuPrime™ PCR kit(Invitrogen)) on 100 ng of the genomic DNA extracted from modified andcontrol cells. Briefly, a 292-bp fragment of the CCR5 locus encompassingthe CCR5-ZFN target site was amplified for 30 cycles (95° C.—30 sec.,60° C.—30 sec., and 68° C.—30 sec.) using the primers C5_Cel_(—)160_F1:AAGATGGATTATCAAGTGTCAAGTCC (SEQ ID NO:29); and C5_Cel_(—)160R1:CAAAGTCCCACTGGGCG (SEQ ID NO:30).

The PCR product was spun through a G-50 column (GE Healthcare) and 1 μlof the purified product was mixed with 1 μl of 10× annealing buffer (1×annealing buffer—10 mM Tris, 100 mM NaCl) and water to a final volume of10 μl. The DNA was denatured and re-annealed in a PCR block using aprogram that allows heteroduplexes to form (95° C.—10 min; 95° C. to 85°C. at −2° C./s; and 85° C. to 25° C. at −0.1° C./s). After re-annealing,10 of the Surveyor™ nuclease (Transgenomics), 1 μl 10× AccuPrime™ PCRbuffer II, and water were added to a total volume of 20 μl. The reactionwas incubated at 42° C. for 20 min to digest heteroduplexes, and thecleaved products were resolved on a non-denaturing 10% TBEpolyacrylamide gel (Bio-Rad). The gel was dried and analyzed using aPhosphorimager®. The level of ZFN-induced target gene disruption wasdetermined by obtaining the ratio of the uncleaved parental fragment tothe two faster-migrating cleaved products. The proportion ofZFN-disrupted CCR5 alleles in the original sample was calculated usingthe formula: (1−√(Parental fraction))×100. The assay is sensitive tosingle nucleotide changes and has a detection limit of ˜1% ZFN-modifiedalleles.

The results, shown in FIG. 13, demonstrated that CCR5-ZFNs as describedherein are highly efficient (50-80%) in mutating CCR5 in GHOST-CCR5cells (lanes 3 and 4). Non-transduced control cells (lane 1) and cellstransduced with an Ad5/35 vector encoding IL-2Rγ-specific ZFNs (Urnov etal. (2005) Nature 435:646-651; lane 2) did not exhibit any detectableCCR5 modification, indicating the results were CCR-5-ZFN specific.

B. HIV Challenge

In addition, the transduced cell populations were maintained in cultureand one week later were infected with HIV-1_(BAL), a prototypeCCR5-tropic HIV-1 isolate. Challenge viruses were obtained from the NIHAIDS Research and Reference Reagent Program and propagated inCD8-depleted PBMC to generate working stocks.

Immediately prior to HIV-1 infection, CCR5 surface expression wasanalyzed and shown to be reduced by >10-fold in the pools of CCR5-ZFNtransduced cells compared to control cells treated with IL2Rγ-ZFNs (FIG.14A).

Results of the HIV-1_(BAL) challenge demonstrated a substantial decreasein HIV-1 infection in CCR5-ZFN treated samples after one week, asmeasured by loss of HIV LTR-driven GFP induction 48 hours afterinfection (FIG. 14B). Genetic modification at the intended target sitewithin CCR5 was confirmed through sequencing of genomic DNA from theCCR5-ZFN treated GHOST-CCR5 cells.

In addition, single cell derived clones isolated from theCCR5-ZFN-transduced GHOST-CCR5 cells were expanded over a period ofseveral weeks. The CCR5 transgene was genotyped and a clone possessingonly disrupted CCR5 alleles was tested and shown to be resistant to HIVinfection by HIV-1BAL. Introduction of a CCR-5 transgene into thesecells restored infectability by HIV, demonstrating that resistance toHIV-1 infection was mediated exclusively by a defect in viral entry viaZFN-mediated CCR5 disruption.

These results show that the CCR5-ZFNs efficiently cleave their DNAtarget site in the CCR5 gene, and confirm that a high proportion ofZFN-induced mutations prevent CCR5 cell-surface expression, resulting incomplete resistance to CCR5-tropic HIV-1 infection.

Example 15 CCR5-ZFN Modification Confers a Survival Advantage

The following experiments were conducted to evaluate if ZFN-mediateddisruption of CCR5 would confer the long-term resistance to HIV-1expected from a permanent genetic change.

A. CCR-5 Disruption after Long-Term Culture

PM1 cells, a CD4⁺ T-cell line with levels of CCR5 expression similar toprimary CD4⁺ T cells, were electroporated with a CCR5-ZFN expressionplasmid encoding the ZFN201 pair to yield an endogenous CCR5 disruptionlevel of 2.4% of the alleles.

This ZFN-treated cell population was then infected with HIV-1_(BAL) ormock infected on day 7, cells were expanded in continuous culture for 70days, and the proportion of ZFN-modified alleles measured by DNAanalysis pre-infection and on days 3, 10, 21, 31, 42 and 52 afterinfection.

As shown in FIG. 15, by day 52 of infection, the HIV-1 infected PM1culture underwent a ˜30-fold enrichment for ZFN-modified CCR5 alleles(˜73%). In contrast, the mock infected population showed stablepersistence of the ZFN-disrupted CCR5 alleles (˜2.3%), indicating noadverse consequences in growth rates for cells carrying a ZFN-modifiedallele in the absence of selective pressure. PM1 cells electroporatedwith control (non-CCR5-targeted) ZFN expression plasmids weresusceptible to HIV-1 infection and showed no evidence of CCR5disruption.

These results demonstrate that HIV-1 infection provides a powerfulselective advantage for CCR5-ZFN modified cells and that the selectiveadvantage is maintained long-term in culture.

B. ZFN-Mediated Mutations

The molecular identity of the ZFN-mediated mutations in the CCR5 gene inthe PM1 cells was also determined by PCR-amplification and sequencing ofthe targeted region of CCR5 at day 52 post-infection.

Numerous molecularly distinct short deletions and insertions in 78% ofsequence reads (63 out of 81 sequences) were observed (FIG. 16),indicating that persistence of modified CCR5 alleles in the presence ofHIV did not result from a single rare event.

All of the mutations mapped at or near the ZFN recognition sites,suggesting the permanent modifications of the CCR5 gene sequenceresulted from ZFN cleavage and subsequent repair via NHEJ. While a broadrange of different deletion and insertion mutations were observed, aspecific 5-bp insertion (a duplication of the sequence between the ZFNbinding sites which results in introduction of two stop codonsimmediately downstream of the isoleucine codon at position 56)represented >30% of all modified sequences (FIG. 16).

C. Superinfection

Superinfection experiments were also conducted to confirm that CCR5-ZFNmodified PM1 cells remained susceptible to CXCR4-tropic HIV-1 andmaintained a selective advantage when re-infected with CCR5-tropicvirus.

Briefly, PM1 cells were mock transfected, or transfected with plasmidsencoding the CCR5-ZFN 201 pair or a control ZFN pair (GR) as describedabove. These cell populations were challenged with HIV-1_(BAL) and onday 59 post-infection a portion of each sample was mixed with parental,non-transfected PM1 cells and re-infected with either CXCR4-tropicHIV-1_(BK132) or CCR5-tropic HIV-1_(BAL). These re-infected cultureswere followed over time and analyzed for gene disruption frequency onday 21 post-reinfection (day 80 post-initial infection). Cells infectedwith HIV-1_(BAL) re-enriched for ZFN-modified cells (64%) followingdilution with the PM1 cells, whereas in cell populations that were mockinfected or infected with the CXCR4-tropic HIV-1_(BK132), little or noselective advantage was observed for CCR5 disrupted cells.

GHOST-CXCR4 cells were also challenged with supernatants (5 μl) fromcultures of HIV-1 challenged CCR5-ZFN transfected PM1 cells removed atearly (day 3) and late (day 56) time points. These cultures showed noCXCR4-dependent infection. The same supernatants applied to GHOST-CCR5cells remained infectious, although to a lesser degree, with theexception of the CCR5-ZFN transfected sample suggesting thatthe >30-fold enrichment for CCR5 null PM1 cells had resulted in greatlyreduced viral infectivity by day 56 of the culture. Thus, viralevolution toward CXCR4 co-receptor usage was not detected insupernatants collected at early and late timepoints from CCR5-ZFNtreated and HIV-1 infected cultures.

In addition, V3 loop sequences were obtained from supernatants of HIV-1challenged PM-1 cells transfected with plasmids expressing eitherCCR5-ZFNs or a GFP control to determine the effects of ZFN generatedCCR5 null cell enrichment on viral tropism over time. 150 proviral HIVDNA sequences were isolated from longitudinal culture of HIV-1_(BAL)infected CCR5 ZFN-treated PM-1 cells; of these, 88 were isolated on day3, and 62 were isolated on day 52 after infection. As a control, 78 HIVDNA sequences were isolated from the HIV infected GFP-treated PM-1cells; 45 at day 3 and 33 on day 52. The sequences were evaluated forchanges in tropism by matching the R5, R5×4, or X4 consensus V3 loopsequences disclosed by Hung et al. (1999) J. Virol. 73:8216-8226. All V3loop sequences from the GFP and CCR5-ZFN treated at both day 3 and day52 samples most closely matched the CCR5 consensus sequence, suggestingno rapid evolution toward switching co-receptor usage; consistent withthe above data showing infectivity in only the CCR5-GHOST reporter cellline.

These results demonstrate that transient expression of CCR5-ZFNsestablishes stable and selective resistance to CCR5-tropic HIV-1,similar to that observed in individuals carrying the naturally occurringCCR5Δ32 mutation.

Example 16 In Vitro Selection of CCR5-ZFN Modified Primary Cd4 T Cells

A. Disruption of CCR5 with ZFNs

To determine the efficacy of CCR5-ZFNs in primary human cells, CD4⁺ Tcells from healthy donors with a wild-type CCR5 gene were transducedwith Ad5/35 vectors encoding either CCR5-ZFNs 215 or CCR5-ZFNs 224 toprovide transient, high efficiency ZFN delivery. Multiplicity ofinfection (MOI)-dependent levels of ZFN-mediated CCR5 disruption(reaching 40-60% of the CCR5 alleles) were observed in multipleexperiments using cells isolated from different donors. An example isshown in FIG. 17.

As shown in FIG. 18, the population-doubling rate of the modifiedprimary CD4 T cells was indistinguishable from that of non-transducedcells, with the proportion of CCR5-modified alleles remaining stable forat least one month during in vitro culture.

B. HIV Challenge

The resistance of bulk ZFN-modified CD4 T cells to HIV infection invitro was also evaluated.

Individuals carrying the naturally occurring CCR5Δ32 mutation have beenshown to be protected from HIV infection and progression. See, forexample, Samson et al. (1996) Nature 382:722-725 (1996); Huang (1996)Nat Med. 2:1240-1243 (1996); Berger et al. (1999) Annu. Rev. Immunol17:657-700. In a control experiment, CD4⁺ T cells from a donorhomozygous for the CCR5Δ32 allele were mixed with CD4⁺ T cells from aCCR5 wild type donor at the indicated ratios, and challenged withHIV-1_(BAL). Following challenge, an ˜2-fold enrichment for CCR5Δ32 CD4T cells, compared to the parallel mock-infected samples, was observed.

Infection of a bulk CCR5-ZFN transduced CD4⁺ T cell population withCCR5-tropic HIV-1_(US1) also resulted in a two-fold enrichment ofgene-edited cells containing ZFN-disrupted CCR5 alleles (measured usingthe Surveyor® nuclease (Cel-1) assay as described above) over 17 days ofculture, while mock-infected control populations maintained a stablelevel of ZFN-disrupted CCR5 alleles (FIG. 19). In parallel experiments,CCR5-ZFN transduced cells challenged with HIV-1_(US1) producedsignificantly lower levels of soluble p24 than controls, consistent withthe frequency of CCR5 disruption in the population. CD4 T cellstransduced with an Ad5/35 GFP control vector showed no detectabledisruption of their CCR5 gene.

Thus, CD4⁺ T cells made CCR5 null via ZFN transduction were selectedwith similar efficiency to CD4 T cells homozygous for the naturallyoccurring CCR5Δ32 allele during HIV-1 infection.

Example 17 Specificity of CCR5-ZFNs in Primary CD4 T Cells

A. Double-Stranded Breaks

To quantify the number of double-stranded breaks (DSBs) generatedpost-ZFN expression, we conducted intranuclear staining for genome-wideDSBs via immunodetection of P53BP1 foci as an unbiased measure of ZFNaction throughout the nucleus. P53BP1 is recruited to the sites of DSBsearly in the repair response and is required for NHEJ (Schultz et al.(2000) J Cell Biol. 151:1381-1390). Briefly, 24 hours post-transductionof CD4⁺ T cells with Ad5/35 vectors expressing CCR5 targeted ZFNs, thenumber of 53BP1 immunoreactive foci per nucleus of the CD4 T cells wasdetermined.

Intranuclear staining for P53BP1 was performed using fixation withmethanol or paraformaldehyde followed by nuclear permeabilization with0.5% Triton. Affinity purified rabbit anti-P53BP1 (Bethel Laboratories)and secondary Alexa Fluor™ 488 F(ab′)2 goat anti-rabbit IgG (H+L)antibody was from Invitrogen. Antibodies were used at 2 to 5 μg/ml finalconcentration. Epifluorescence microscopy was performed using a ZeissAxioplan-II (Thornwood N.Y.) with a Zeiss 63x Plan Apo objective havinga numerical aperture of 1.4.

Images were acquired and analyzed using Improvision Volocity™ softwarepackage (Lexington Mass.) acquisition and classification modules.Analysis of discrete regions of P53BP1 fluorescence was performed byadjusting exposure time and thresholds to minimize autofluorescence andby intensity gating to include the top 40% of fluorescence. Individualregions identified were then enumerated and measured. Only greenfluorescent regions that colocalized with DAPI fluorescence wereincluded in final analyses.

Results are shown in FIG. 20. There was no significant difference in themean number of intranuclear P53BP1 foci when comparing non-transducedand ZFN 224 transduced CD4 T cells. In contrast, etoposide-treatedpositive control cells (p=0.004) or cells transduced with an Ad5/35vector expressing ZFN 215 (p=0.003) showed a statistically significantelevation in P53BP1 intranuclear foci when compared to non-transducedcells. In addition, no significant difference in the mean perimeter ofp53BP1 foci was observed among all conditions. DNA analysis confirmedequivalent degrees of cleavage at the intended target site in the CCR5gene by both ZFN 215 and ZFN 224.

B. Determination of the Consensus ZFN Binding Site

To confirm the specificity of ZFN 224 action, the consensus ZFN bindingsites were determined and found to match the unique intended targetsequence in CCR5. Binding site preferences for each of the 2 zinc fingerproteins comprising ZFN-224 were assayed using a site selection methodas follows: (1) first, an HA-tagged version of the ZFP of interest wasexpressed via the TnT quick coupled transcription-translation system(Promega), and incubated with a pool of partially randomized DNAsequences in the presence of biotinylated anti-HA Fab fragments (Roche)and poly dIdC competitor DNA (Sigma); (2) the protein—along with anyproductively bound DNA sequences—was captured on streptavidin coatedmagnetic beads (Dynal); (3) the magnetic beads were placed in Roche PCRmaster mix containing the appropriate primers and the bound DNA was thenreleased and PCR amplified. This amplified pool of DNA was then used asthe starting DNA pool for subsequent rounds of ZFP binding, enrichmentand amplification. Cycles comprising steps (1)-(3) were repeated for atotal of four rounds of selection. Finally, DNA fragments amplifiedafter the final round were cloned and sequenced. The randomized regionof each DNA sequence was aligned to determine the consensus binding sitesequence for the zinc finger DNA binding domain. The consensus bindingsites determined by this method agreed with the binding sites specifiedin Table 1.

Target sequence preferences for the two CCR5 ZFNs of the ZFN 224 pair(8196z and 8266, Table 1), determined as described above, were used toguide a genome wide bioinformatic prediction of the top 15 potentialoff-target, sites in the human genome. This bioinformatic analysissearched for and ranked the potential off-target sites as follows:

All potential DNA binding sites for the two members of the ZFN224 pair(8196z and 8266, Table 1) were identified in the human genome, allowingfor up to two base pair mismatches from the consensus sequences of eachtarget site determined as described above.

All possible cleavage locations were identified using the complete listof binding sites (identified as described in the previous paragraph)that allowed any two ZFNs (including homodimerization andheterodimerization events) to bind in the appropriate configuration fornuclease activity (i.e. ZFNs binding on opposite sides of the DNA witheither a 5 or 6 bp spacing between them).

The resulting list of potential cleavage sites was then ranked to givepriority to those sites with the highest similarity to the consensus foreach ZFN as defined by the site selection method described above.Briefly, the site selection data was used to create a probability forthe recognition of all four nucleotides (A, C, G or T) in each of the 12positions of the binding site for each ZFN. Each putative ZFN bindingsite was scored as the product of these twelve (12) probabilities. (Notethat to eliminate a score or probability of zero every position had asingle count added for each nucleotide (A, C, G or T) prior tonormalization to ensure no entry in the probability table was zero).Similarly, the score for a given off-target cleavage site (requiring twosuch ZFN sites to be occupied) was calculated as the product of the twoscores given to each of the two ZFN binding sites comprising theputative cleavage site. Of the 15 sites identified, 7 fall withinannotated genes and 2 of these fall within exonic sequence. These sevengenes share the following characteristics; (i) their mutation ordisruption has not been connected with any known pathology; and (ii)with the exception of CCR2, they have no described function in CD4T-cells.

Surveyor™ nuclease assays revealed no detectable ZFN activity (1% limitof detection) at any of these sites with the exception of CCR2 (theclosest relative of the CCR5 gene in the human genome). We observed 4.1%modification of CCR2 alleles in the population under conditions thatrevealed 35.6% ZFN-modified CCR5 alleles. However, loss of CCR2 in CD4 Tcells should be well tolerated since CCR2−/− mice display numerous mildphenotypes predominantly associated with delayed macrophage traffickingand recruitment (Peters et al. (2000) J. Immunol. 165:7072-7077). Mutantalleles of CCR2 have been correlated with delayed progression to AIDS inHIV infected individuals, although no influence on the incidence ofHIV-1 infection was observed (Smith et al. (1997) Nat. Med.3:1052-1053). Thus, parallel mutation of CCR2 is unlikely to bedeleterious and may increase protection of modified CD4 T cells to HIVinfection.

The combination of ZFP consensus binding site-directed analysis of themost similar off-target sites in the genome with the unbiasedintranuclear staining for genome-wide DSB generation indicates that ZFN224 is a highly specific engineered nuclease with measurable activityonly at the CCR5 gene and, to a ˜10-fold lesser extent, at the CCR5homologue CCR2.

Example 18 In Vivo Selection of CCR5-ZFN Modified Primary CD4 T Cells

A NOG/SCID mouse model of HIV infection was used to test adoptivetransfer and protection from HIV infection of the ZFN-modified CD4 Tcells in vivo. See Schultz et al. (2007) Nat. Rev. Immunol. 7:118.

Primary CD4 T cells were transduced with the Ad5/35 vectors and expandedin culture using anti-CD3/anti-CD28 coated magnetic beads in thepresence of IL-2. NOG/SCID mice (7-9 weeks old) were randomly assignedto 2 treatment groups (n=8 mice per group) with equal mix of males andfemales in each group. These mice were maintained in a defined floraanimal facility. Both groups received an IP injection of 100 μl of PBScontaining 7.5 million CCR5-ZFN ex-vivo expanded primary human CD4 Tcells and 1 million resting, autologous PBMCs to promote engraftment incombination. In addition, the mock treated animals received 1 millionnon-infected PHA-activated autologous PBMCs, whereas the infected groupof animals received 1 million CCR5-tropic HIV-1_(US1) infectedPHA-activated PBMCs.

To assess engraftment, peripheral blood sampling was performed three andfour weeks after adoptive transfer and analyzed for engraftment by flowcytometry for human CD45, CD4 and CD8. After 4.5 weeks, mice weresacrificed and splenic CD4 T lymphocytes were purified using MiltenyiMACS separation kit. Only samples with greater than 75% purity were usedfor the final analysis. To determine CCR5 disruption frequency, amodified Surveyor™ nuclease assay was employed by utilizing a nested.PCR approach to fully remove contaminating mouse genomic DNA. The DNAfrom purified splenic CD4 cells was amplified first using 50 pmols ofoutside primers (R5-det-out-F1: CTGCCTCATAAGGTTGCCCTAAG (SEQ ID NO:31);C5_HDR_R: CCAGCAATAGATGATCCAACTCAAATTCC (SEQ ID NO:32)) for 25 cycles(95° C.—30 sec., 58° C.—30 sec., and 68° C.—3 min.), the resultingmaterial was gel purified, and the Surveyor™ nuclease assay wasperformed on the purified product as per the manufacturers'recommendations.

After a month of HIV infection in vivo, mice were sacrificed and genomicDNA from human CD4 T lymphocytes purified from the spleen was used foranalysis of ZFN-mediated CCR5 disruption, using the Surveyor™ nucleaseassay described above. Samples from 2 mice (one HIV-infected and onemock-infected) were excluded from analysis due to inadequate CD4 cellpurification:

All groups showed equal engraftment, although the HIV infected groupsexhibited a reduced CD4 to CD8 T cell ratio, consistent with HIV-inducedCD4 T cell depletion.

Further, FIG. 21 shows that an approximately 3-fold enrichment forZFN-disrupted CCR5 alleles was observed in the HIV infected group (27.5%average CCR5-disruption), compared to animals receiving an identicalstarting population of ZFN-treated CD4 T cells in the absence of HIVinfection (mock group, 8.5% average CCR5 disruption, p=0.008).

These data demonstrate (i) a selective advantage for ZFN-transducedprimary human CD4⁺ T cells in the presence of HIV-1 in vivo, and (ii)normal engraftment and growth of these same ZFN-transduced cells even inthe absence of this selective pressure. These data indicate that thetransient delivery of engineered ZFNs succeeded in reproducing theCCR5Δ32 null genotype (and resulting phenotypes).

Thus, ZFNs as described herein cleave specifically in the CCR5 gene, andcause permanent disruption of greater than 50% of the CCR5 alleles in abulk population of primary human CD4⁺ T-cells. In addition, the geneticdisruption of CCR5 by ZFNs provides robust, stable, and heritableprotection against HIV-1 infection in vitro and in vivo. ZFN-modifiedCD4 T-cells engraft and proliferate normally upon stimulation.

1. A protein comprising an engineered zinc finger protein DNA-bindingdomain, wherein the DNA-binding domain comprises four zinc fingerrecognition regions ordered F1 to F4 from N-terminus to C-terminus, andwherein F2, F3, and F4 comprise the following amino acid sequences:(SEQ ID NO: 17) F2: QKINLQV (SEQ ID NO: 12) F3: RSDVLSE (SEQ ID NO: 13)F4: QRNHRTT.


2. The protein according to claim 1, wherein F1 comprises the amino acidsequence RSDNLGV (SEQ ID NO:16).
 3. The protein according to claim 1,wherein F1 comprises the amino acid sequence RSDNLSV (SEQ ID NO:10). 4.A protein according to claim 1, further comprising a cleavage domain. 5.The protein of claim 4, wherein the cleavage domain is a cleavagehalf-domain.
 6. The protein of claim 5, wherein the cleavage half-domainis a wild-type FokI cleavage half-domain.
 7. The protein of claim 5,wherein the cleavage half-domain is an engineered FokI cleavagehalf-domain.
 8. A polynucleotide encoding the protein of claim
 1. 9. Agene delivery vector comprising a polynucleotide according to claim 8.10. The gene delivery vector of claim 9, wherein the vector is anadenovirus vector.
 11. The gene delivery vector of claim 8, wherein theadenovirus vector is an Ad5/35 vector.
 12. An isolated cell comprisingthe protein of claim
 1. 13. The isolated cell of claim 12, furthercomprising a protein comprising an engineered zinc finger proteinDNA-binding domain, wherein the DNA-binding domain comprises four zincfinger recognition regions ordered F1 to F4 from N-terminus toC-terminus, and wherein F1, F3, and F4 comprise the following amino acidsequences: (SEQ ID NO: 2) F1: DRSNLSR (SEQ ID NO: 4) F3: RSDNLAR(SEQ ID NO: 8) F4: TSGNLTR.


14. An isolated cell comprising the polynucleotide of claim
 8. 15. Theisolated cell of claim 14, further comprising a polynucleotide encodinga protein comprising an engineered zinc finger protein DNA-bindingdomain, wherein the DNA-binding domain comprises four zinc fingerrecognition regions ordered F1 to F4 from N-terminus to C-terminus, andwherein F1, F3, and F4 comprise the following amino acid sequences:(SEQ ID NO: 2) F1: DRSNLSR (SEQ ID NO: 4) F3: RSDNLAR (SEQ ID NO: 8)F4: TSGNLTR.


16. The cell of claim 12, wherein the cell is selected from the groupconsisting of a hematopoietic stem cell, a T-cell, a macrophage, adendritic cell and an antigen-presenting cell.
 17. The cell of claim 16,wherein the T-cell is a CD4⁺ cell.
 18. The cell of claim 14, wherein thecell is selected from the group consisting of a hematopoietic stem cell,a T-cell, a macrophage, a dendritic cell and an antigen-presenting cell.19. The cell of claim 18, wherein the T-cell is a CD4⁺ cell.
 20. Amethod for inactivating the CCR-5 gene in a human cell, the methodcomprising administering to the cell a polynucleotide according to claim8.
 21. The method of claim 20, further comprising administering apolynucleotide encoding a protein comprising an engineered zinc fingerprotein DNA-binding domain, wherein the DNA-binding domain comprisesfour zinc finger recognition regions ordered F1 to F4 from N-terminus toC-terminus, and wherein F1, F3, and F4 comprise the following amino acidsequences: (SEQ ID NO: 2) F1: DRSNLSR (SEQ ID NO: 4) F3: RSDNLAR(SEQ ID NO: 8) F4: TSGNLTR.


22. The method of claim 20, wherein the cell is selected from the groupconsisting of a hematopoietic stem cell, a T-cell, a macrophage, adendritic cell and an antigen-presenting cell.
 23. The method of claim22, wherein the T-cell is a CD4⁺ cell.